The aim of this application is to develop an optimized prime-boost vaccine regimen to HIV-1 antigens in a mouse model. Specifically, we will use E1-deleted adenoviral (Ad) recombinants of the recently vectored simian serotypes 6 and 68 in comparison to the human serotype 5 expressing gag of HIV-1 to assess the efficacy of homologous versus heterologous Ad prime boost regimens on the magnitude and the breadth of the transgene product-specific CD8+ T cell response. The simian Ad vectors were developed to circumvent interference due to neutralizing antibodies to common serotypes of human Ad virus, such as serotype 5, that are present in a sizable percentage of the human population. Ad prime-boost vaccination will be compared with DNA vaccine prime-Ad boost immunizations. Our data generated thus far indicate that heterologous Ad prime boosting induces maximal CD8+ T cell frequencies, provided that the 2nd immunization is not applied until several months later. Our data also indicate that the magnitude of the CD8+ T cell response is influenced by the order of the use of the different Ad vaccine carriers. Specific emphasis will thus be given to determine the optimal time interval between the 1st and the 2nd immunization and the most efficacious sequence of vaccine carriers used for either vaccination. In the second part of this application, we propose to elucidate the immunological pathways that dictate the lengthy time interval between the 1st and 2nd immunizations. We will test the hypothesis that the antigens encoded by the Ad vectors, especially the simian Ad vectors, persist for a drawn-out period of time resulting in an extended T cell effector phase and a delayed memory phase, which in turn has a negative impact on a pre-mature booster immunization. Persistence of vector encoded antigen upon Ad immunization could potentially reflect persistence of the vector, which would have implications on the safety of Ad vaccines. Persistence of the transgene product on the other hand may be exceedingly useful for a vaccine to HIV-1 where a long-lasting effector CD8+ T cell population would be expected to provide superior protection compared to memory CD8+ T cells.